NADH:ubiquinone oxidoreductase (complex-I) is the most common site of impairment of the oxidative phosphorylation-related disorders. These disorders include some neurodevelopmental and neurodegenerative syndromes such as Leigh syndrome, MELAS, and some forms of Parkinson's disease. While the clinical symptoms of complex-I deficiency are defined, signaling pathways regulating complex-I function in the mitochondria are less understood. Recently, our group found that unregulated glycogen synthase kinase-3[unreadable] (GSK3[unreadable]) activity inhibits complex-I function, increases reactive oxygen species production, fragments mitochondria, and increases the cell's sensitivity to complex-I toxins. GSK3[unreadable] is a signaling protein that is known to affect metabolic pathways and brain development. Its regulation is multi-tiered meaning that its actions can be controlled by phosphorylation at its serine-9 site, by protein-protein interactions, and by its intracellular localization. Interestingly, no one has fully elucidated its intracellular distribution in the brain. In preliminary results, we have found that a salient fraction of GSK3[unreadable] exists in brain mitochondria. Furthermore, we have now found previously undiscovered pockets of GSK3[unreadable] expression in other cellular compartments. The first specific aim of this proposal is to fully determine the neuroanatomic ultrastructural distribution of GSK3[unreadable] in the mouse and human brain by electron microscopy. A thorough investigation of this sort has not been conducted previously. The actions of endogenous GSK3[unreadable] signaling in the mitochondria are not fully known. In specific aim 2, the goal is to test the hypothesis that endogenous GSK3[unreadable] modulates complex-I functions. Endogenous mitochondrial GSK3[unreadable] activity is manipulated by molecular and pharmacological methods to assess the affects of GSK3[unreadable] signaling on complex-I. The proteins STAT3 and GRIM19 are bound together in the mitochondria and both are involved in complex-I function. It is also known that GSK3[unreadable] associates with both these proteins. Our hypothesis is that GSK3[unreadable] signaling regulates complex-I activity through a tripartite protein composite consisting of GSK3[unreadable], STAT3, and GRIM19. With this project we hope to elucidate mitochondrial GSK3[unreadable] signaling and its affects on complex-I with the prospect that our findings could be used to discover measures to alleviate complex-I disorders. PUBLIC HEALTH RELEVANCE: This project investigates the intracellular localization and functions of mitochondrial GSK3[unreadable], a protein whose unregulated activity has been previously implicated in severe mitochondrial deficiencies. In humans, mitochondrial dysfunctions have major ramifications on neurodevelopment and neurodegenerative diseases in both infants and adults.